Method for detecting mild impaired glucose tolerance or insulin hyposecretion

ABSTRACT

It is intended to provide a noninvasive method of conveniently detecting mild impaired glucose tolerance and/or insulin hyposecretion at the early stage with the use of an enzyme. Namely, mild impaired glucose tolerance and/or hyposecretion at the early stage are detected by quantifying myoinositol secreted into the urine before loading glucose and after loading glucose for a definite period of time with the use of a reagent and comparing the increase (or the increase ratio) in the myoinositol content thus measured with a characteristic level which has been preliminarily determined in normal subjects.

FIELD OF THE INVENTION

The present invention relates to a method of examining mild impairedglucose tolerance or insulin secretory defect using a sample such asurine. In addition, the present invention can be applied to a method forpredicting or diagnosing a disease that stems from mild impaired glucosetolerance or insulin secretory defect, such as diabetes meritus,arteriosclerosis, or hypertension; a method of determining effects ofprevention of, treatment of, or medical advice on those diseases; and amethod of evaluating therapeutic agents for treatment of those diseases.

BACKGROUND OF THE INVENTION

A final goal of diabetic treatment is to prevent the onset of diabeticcomplications and to inhibit the development thereof As demonstrated byclinical tests for achieving this goal it is important to find anyabnormality and start treatment thereof at the earliest possible stage[e.g., Diabetes Research and Clinical Practice, 28, 103 (1995)].

Further, it is considered effective as a more advanced preventive methodto find individuals with prediabetes or at prestage of diabetes, orindividuals with mild impaired glucose tolerance or insulin secretorydefect, who are not prediabetic at present but are highly likely todevelop diabetes or prediabetes in the near future, and give themtreatment or advice for exercise and dietary. Clinical tests have beenconducted to scientifically demonstrate this [e.g., Diabetes Care, 21,1720 (1998)]. Therefore, detecting individuals with prediabetes will beimportant for prevention of diabetes mellitus and also complicationsthereof. Furthermore, diagnosing individuals with mild impaired glucosetolerance or insulin secretory defect, who are not prediabetic atpresent but are highly likely to develop diabetes or prediabetes in thenear future, is considered most important for purpose of preventingdiabetes mellitus at an earlier date.

An example of the diagnostic method for diabetes mellitus is an oralglucose tolerance test. After a 75 gram oral glucose load, a group ofindividuals with the fasting blood glucose level being less than 110mg/dl and the 2-hour postload blood glucose level being less than 140mg/dl is defined as normal glucose tolerance (NGT). In addition, a groupof individuals with the fasting blood glucose level being not less than110 mg/dl but less than 126 mg/dl and the 2-hour postload blood glucoselevel being less than 140 mg/dl is defined as impaired fasting glycemia(IFG); and a group of individuals with the fasting blood glucose levelbeing less than 126 mg/dl and the 2-hour postload blood glucose levelbeing not less than 140 mg/dl but less than 200 mg/dl is defined asimpaired glucose tolerance (IGT); and both groups IFG+IGT are defined asborderline type. A group of individuals with the fasting blood glucoselevel being not less than 126 mg/dl or the 2-hour postload blood glucoselevel being not less than 200 mg/dl is defined as diabetes meritus.

The guideline of the Japan Diabetes Society teaches that amongindividuals defined as NGT on the basis of only the fasting bloodglucose level and the 2-hour postload blood glucose level, those withthe 1-hour postload blood glucose levels being 180 mg/dl or more arex athigher risk to develop diabetes so that they should be handled as theborderline type.

The term “impaired glucose tolerance” or “glucose tolerance failure”refers to the condition of an increase in blood glucose level caused byinsufficient uptake of blood glucose into peripheral tissues such asskeletal muscle, liver, and adipocyte after glucose is introduced intothe blood through meals. In addition, the term “mild impaired glucosetolerance” refers to that the increment is slightly higher than that ofhealthy individuals.

Insulin is a hormone secreted from beta cells of pancreas and acts onskeletal muscle, liver and adipose tissue to lower the blood glucoselevel. The term “insulin secretory defect” refers to the condition ofinsufficient insulin secretion to uptake a sufficient amount of bloodglucose into peripheral tissues such as skeletal muscle, liver, andadipocyte after glucose is introduced into the blood through meals orthe like. Among the insulin secretory defect, the condition ofinsufficient insulin secretion to uptake the blood glucose intoperipheral tissues just after glucose is introduced into the blood isreferred to as “impaired early insulin secretion”. According to theguideline of the Japan Diabetes Society, the term “impaired earlyinsulin secretion” refers to the condition in which the insulinogenicindex I.I is less than 0.4. Insulinogenic index I is defined as ΔIRI(30-0)/ΔPG (30-0) wherein ΔIRI (30-0) means between the differencebetween the blood insulin levels at 30 min after glucose load and beforeglucose load; and APG (30-0) means the difference between the bloodglucose levels at 30 min after glucose load and before glucose load.

Assays of blood glucose levels and insulin levels for those diagnosesare invasive procedures that require blood drawing more than once withina short time, giving the subjects considerable pains. Therefore, thereis a need for a simple assay with lower invasiveness, which can solvethese disadvantages, preferably a noninvasive assay.

On the other hand, the quantitative determination of myo-inositol in abiological sample has been considered useful for the diagnosis ofdiabetes mellitus and the following reports have been provided.

(a) In diabetes mellitus, there was an increase in the urinarymyo-inositol level [Larner J. et al., New Eng. J. Med., 323, 373-378(1990)].

(b) No difference was found between NGT and the borderline type withrespect to the urinary myo-inositol level [Susumu Suzuki, Diabetes Care,Vol. 17, No. 12 (1994) 1465-1468].

(c) The borderline type (IFC; IGT) and diabetes meritus showed higherurinary myo-inositol level than that of NGT (JP 2001-190299 A).

The above reports (a) and (b) show the results obtained by determiningthe urinary myo-inositol levels with GC/MS. Nevertheless, the data areproblematic in reproducibility and reliability because they varied amongdifferent examiners. On the other hand, in the report (c), the resultsare more precise and reliable than those obtained by GC/MS because theyare obtained by determining the urinary myo-inositol level with ahigh-sensitive myo-inositol assay reagent using an enzyme. In this way,the detection of the group with prediabetes has become possible.

However, the myo-inositol assay reagent used in the report (c) hasproblems including: (i) an insufficient lower limit of detection becauseof a narrow measurement range and the need of diluting a sample fordetermination of various urinary myo-inositol levels; and (ii)insufficient avoidance of effects of coexisting substances in urine,particularly glucose. Therefore, the detection of mild impaired glucosetolerance and insulin secretory defect, which fall within NGT, has beenimpossible.

Furthermore, if a subject is judged to be NGT on the basis of only bloodlevels before a 75 g oral glucose load and at 2 hours after the glucoseload, such judgment does not reflect the change in blood level from 0 to2 hours. For example, even individuals (with mild impaired glucosetolerance and insulin secretory defect) who keep a higher blood glucoselevel from just after the glucose load, and thus are highly likely todevelop diabetes or prediabetes in the near future are practicallyclassified in NGT. As used herein, the term “mild impaired glucosetolerance” is classified in NGT, but refers to a slight decrease inglucose tolerance characterized by, when the loading test is carried outand blood samples are collected four times on fasting and at 30 minutes,1 hour, and 2 hours after the load, (i) an oxyhyperglycemia, i.e., veryhigh blood glucose levels (180 mg/dL or more) at 30 minutes and 1 hourafter the load, (ii) a higher blood glucose level than that of healthyindividuals at 2 hours after the load although the blood level is lessthan 140 mg/dL (e.g. not less than 120 mg/dL), (iii) high ΣPG (e.g. 530mg/dL or more), i.e. the total of blood glucose levels just before 75 goral glucose load and at 30, 60, and 120 minutes after the glucose loadTherefore, it can not be anticipated from the public disclosures (a)-(c)to identify the group of individuals who are highly likely to developdiabetes or prediabetes in the near future, for example, individualswith mild impaired glucose tolerance, by determining myo-inositollevels.

In this way, the conventional technology teaches no method of detectingmild impaired glucose tolerance and insulin secretory defect, which keepa higher blood glucose level from just after the glucose load, and thusare highly likely to develop to diabetes or prediabetes in the nearfuture.

SUMMARY OF THE INVENTION

The present invention intends to provide an assay method for simpledetermination of mild impaired glucose tolerance and/or insulinsecretory defect with good reproducibility.

For achieving this object, the present inventors considered that searchfor any marker for effectively determining mild impaired glucosetolerance and/or insulin secretory defect was advantageous. As a resultof concentrated efforts, whereas myo-inositol is conventionallyconsidered to be useful for detection of insulin resistance andprediabetes (borderline type and diabetes mellitus), the presentinventors unexpectedly found that myo-inositol is also useful as amarker for effectively detecting mild impaired glucose tolerance orinsulin secretory defect

Blood serum, plasma, or urine collected from the human body, or ahomogenized extract of living tissue are used as a sample. Urine ispreferable because it can be non-invasively obtained.

The present inventors continued to develop a high-sensitive quantitativedetermination assay of myo-inositol and a composition for the assay toprovide a simple and cost-effective quantitative determination assay ofmyo-inositol with a high degree of accuracy (JP 06-61278 B). Thisenzymatic assay, which does not require any preliminary treatment,opened the way to obtain reliable data of myo-inositol for the firsttime. Such development of the high-sensitive quantitative determinationassay and the composition for the quantitative determination allowed thefirst success of providing the method of the present invention forexamining mild impaired glucose tolerance and/or insulin secretorydefect.

Furthermore, after the administration of a given amount of glucose to asubject, urine samples were obtained non-invasively from the subjectwithin a given time period and the myo-inositol levels thereof weredetermined using the myo-inositol assay reagent as described above. Thedetermination revealed that not only individuals with prediabetes (ofborderline type, IFC IGT) and individuals of diabetes mellitus, but alsoindividuals practically showing mild impaired glucose tolerance in spiteof being of NGT and individuals practically showing a decrease in earlyinsulin secretion in spite of being of NGT have higher levels than thecharacteristic value predetermined from healthy individuals. Therefore,it has been found that the assay reagent of the present inventionenables not only the distinction between NGT and non-NGT with progressedimpaired glucose tolerance (borderline type, IF4 IGT, diabetes) but alsothe simple, highly reproducible and efficient distinction of individualspractically showing mild impaired glucose tolerance in spite of being ofNGT or individuals practically showing a decrease in early insulinsecretion in spite of being of NGT from healthy individuals.

In addition, the concentration of myo-inositol in a sample may be verylow and some of myo-inositol dehydrogenases used may react weakly withglucose. Thus, the elimination of glucose may be required in advance. Amethod for the elimination of glucose includes one using extremechemical stability of myo-inositol and one by modifying glucose using anenzyme as a catalyst. The method using the chemical stability includes,for example, one by heating a sample in the presence of 6 N HCl to allowthe acid decomposition of sugars except myo-inositol and recoveringmyo-inositol remained in the decomposed product; and one by treating asample with a reducing agent such as sodium borohydride to reduce sugarshaving carbonyl groups or formyl groups such as glucose exceptmyo-inositol and modifying them to make compounds unreactive withmyo-inositol dehydrogenase, i.e. an enzyme for the quantitativemyo-inositol assay. The method by modifying glucose using an enzyme as acatalyst includes one by converting glucose in a sample into gluconicacid with glucose oxidase (EC1,1,3,4) and one by converting glucose in asample into glucosephosphate with hexokinase (EC2,7,1,1).

Various improvements are known in these converting methods. In relationto the method of converting glucose into gluconic acid with glucoseoxidase, for example, known is a method to eliminate hydrogen peroxideproducts by catalase after the reaction with glucose oxidase (JP63-185397 A).

Furthermore, in relation to the method of converting glucose intoglucose-6-phosphate with hexokinase, there are known methods to convertglucose into fructose-1,6-bisphosphate using phosphohexose isomerase and6-phosphofructokinase to prevent glucose-6-phosphate from beingreconverted into glucose through an equilibrium reaction (JP 05-76397A); to perform the reaction with glucose-6-phosphate dehydrogenase inthe presence of an oxidized coenzyme (JP 01-320998 A, JP 03-27299 A);and to perform the reaction with pyruvate kinase in the presence ofadenosine diphosphate to prevent the change of adenosine triphosphatelevel decreasing as glucose is eliminated and thus keep the adenosinetriphosphate level constant (JP 02-104298 A).

However, when glucose is eliminated using hexokinase, the enzymaticreaction produces a large amount of ADP in the reaction solution, sothat the effect thereof on the enzymatic reaction cannot be ignored.Thus, it is preferable to convert the resulting ADP to a compound thatdoes not affect the reaction.

Thus, the present inventors have considered an effective method using anADP eliminating agent to convert ADP generated in the reaction solutionby the enzymatic reaction to a compound that does not affect thereaction.

Any substances capable of converting ADP to a compound that does notaffect the reaction can be used as an ADP eliminating agent Of these,enzymes are preferable and kinases that catalyze conversion of ADP toAMP are more preferable. Kinases are also called phosphokinases orphosphotransferases. Examples of the kinases which catalyze conversionof ADP to AMP include pyrophosphate-glycerol transferase,6phosphofructokinase, acetate kinase, and ADP-hexokinase.

As a result of keen investigations, the present inventors have foundthat 6phosophofructokinase and ADP-hexokinase are preferably used as anADP eliminating agent in the present invention.

When 6phosphofructokinase is used as an ADP eliminating agent in thereaction of eliminating glucose in a sample, ADP is produced along withthe conversion of glucose to glucose-6phosphate using ATP-hexokinase inthe presence of ATP and is simultaneously reacted with6phosphofructokinase to allow the conversion of ADP to AMP along withthe conversion of preadded fructose-6phosphate tofructose-1,6bisphosphate.

When ADP-hexokinase is used as an ADP eliminating agent in the reactionof eliminating glucose in a sample, ADP is produced along with theconversion of glucose to glucose-6phosphate using ATP-hexokinase in thepresence of ATP and can be converted to AMP.

In addition, it is preferable to perform such reaction in the presenceof salts. Examples of the salts include: magnesium salts such asmagnesium chloride and magnesium acetate; and potassium salts such aspotassium chloride and potassium sulfate. The concentration of saltsused is, but not limited to, preferably about 1 to 100 mM.

Any of these compounds produced by the enzymatic modification are notreactive with myo-inositol dehydrogenase, an enzyme for the quantitativemyo-inositol determination. The present inventors have found that it ismore preferable to previously eliminate glucose by these methods.

Further, the present inventors have found that myo-inositol can bedetermined more accurately when two kinds of kinases, ATP-hexokinase andADP-hexokinase, are used simultaneously because the influence of sugarsin a sample is reduced. In addition, the present inventors have foundthat the range of myo-inositol determination can be extended by about 10times by adjusting thio-NAD level to a final concentration of 0.1 mM ormore, preferably 2 to 10 mM. Thus, the present inventors have completeda higher sensitive assay system.

The term “characteristic value” refers to a value determined on thebasis of an average of myo-inositol levels in urine samples of healthysubjects selected from those of NGT; standard deviation; and ROC(response operating characteristic) curve. When urine samples are used,the increment in urinary myo-inositol excretion between before theglucose load and at a predetermined time after the glucose load is inthe range of 0 to 20 μg/mg creatinine; or 5 to 15 μg/mg creatinine; ormore preferably 8 to 12 μg/mg creatinine. In addition, thecharacteristic value may be changed if a large-scale examination isconducted in the future and the determination is conducted for healthyindividuals selected clinically. Furthermore, the characteristic valuemay also vary depending on the selected populations of race, sex, andage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of study on thio-NAD levels according toReference Example 1.

FIG. 2 shows the results of stability test of myo-inositol assayreagents according to Reference Example 2.

FIG. 3 shows the effects of ADP-hexokinase according to ReferenceExample 3.

FIG. 4 shows the calibration curve for myo-inositol levels according toReference Example 4.

FIG. 5 shows the relationship between myo-inositol level and ΣPGaccording to Example 1.

FIG. 6 shows the relationship between inositol level and insulinogenicindex according to Example 2.

FIG. 7 shows the relationship between each group and ΣPG according toExample 3.

FIG. 8 shows the relationship between each group and insulinogenic indexaccording to Example 4.

FIG. 9 shows the relationship between each group and myo-inositol levelaccording to Example 5.

FIG. 10 shows the relationship between each group and myo-inositol levelaccording to Example 6.

FIG. 11 shows the correlation between urinary myo-inositol levels in theglucose tolerance test and in the meal tolerance test according toExample 7.

FIG. 12 shows the relationship in each group between Δ myo-inositol inthe glucose tolerance test and Δ myo-inositol in the meal tolerance testaccording to Example 8.

FIG. 13 shows the relationship in urinary glucose negative individualsbetween urinary myo-inositol level in the meal tolerance test and mildimpaired glucose tolerance according to Example 9.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention and preferred embodiments thereof will bedescribed in more detail as below.

According to the present invention, the detection of mild impairedglucose tolerance and insulin secretory defect is carried out bydetermining the amounts of myo-inositol excreted in urine of a subjectbefore the glucose load and at a predetermined time after the glucoseload using the reagent of the present invention; and making a comparisonof an increasing amount or increasing rate of myo-inositol betweenbefore and after the glucose load with the characteristic value definedin advance for healthy individuals.

The increasing amount is calculated as a difference between themyo-inositol content at a predetermined time after the glucose load andthe myo-inositol content before the glucose load, and the increasingrate is calculated as a ratio of the myo-inositol content at apredetermined time after the glucose load to the myo-inositol contentbefore the glucose load.

For the concentration of myo-inositol, an actually determined value maybe used, or a relative value with respect to an appropriate standardindex may be used for compensating the dilution of urine with drinkingwater. Preferably, the index is a urinary creatinine level. The subjectsinclude all individuals in addition to those suspected of beinglifestyle-related diseases such as diabetes.

Any amounts of glucose loaded and any types of the glucose loadingmethods may be used. However, preferable is an oral administration of anaqueous 75 g glucose solution as used in a typical glucose load test ora meal ingestion.

Urine samples may be collected before the glucose load and at any timesuntil 6 hours passed just after the glucose load, preferably at 30minutes to 3 hours after the glucose load. A urine collection period issuitably selected from 30 minutes to 3 hours.

When using urine as a sample, it is collected by a non-invasive method,so that there is no need to select the sampling method, time, and place.For instance, such a sample can be easily prepared by the subject athome, office, school, or the like, and the collected urine sample may betransported directly or in a form of urine-immersed filter paper orother suitable forms, these eliminating the need to be tied to medicalinstitutions or the like. Thus, the present invention provides aprominent method in which, when a filter paper or the like isimpregnated with urine, the sample dispatched is extracted by a suitablemethod and provided to the simple and rapid assay of the presentinvention and then immediately the results is sent to the subject.

In particular, urinary myo-inositol levels can be monitored as neededwhile the subject spends everyday life as usual without glucose load.For example, it is possible to grasp the degree of impaired glucosetolerance or the degree of insulin secretory defect using the maximummyo-inositol level or the difference between the maximum myo-inositollevel and the minimum myo-inositol level in a day. In addition,monitoring urinary myo-inositol levels as needed allows the subject toreconsider the diet contents and control the amount of exercise toprevent diabetes or the progress thereof while having a regular life.

The methods of monitoring urinary myo-inositol levels include any typesof methods capable of detecting myo-inositol, for example, a methodusing a test paper onto which an enzyme that acts on myo-inositol isfixed and a method of electrochemically detecting myo-inositol using anelectrode as a sensor onto which an enzyme acting on myo-inositol isfixed.

In the test paper method, for example, hydrogen peroxide is generated byoxidase and reacted to peroxidase to generate active oxygen, and theactive oxygen causes the oxidation of chromogen for coloration, theintensity of which may be observed. The chromogen includes, but notlimited to, potassium iodide, tetramethylbenzidine,N-(3-sulfopropyl)3,3′,5,5′-sodium tetramethylbenzidine,4-aminoantipyrine, and O-tolidine.

For detecting by means of the sensor, for example, when oxidase is used,hydrogen peroxide generated may be directly measured using an electrode;or the oxidation-reduction current obtained through an electron carriersuch as a ferrocene derivative or a quinone derivative or the quantityof the electric current may be measured Likewise, when dehydrogenase isused, the reduced coenzyme may be directly measured using an electrode;or the oxidation-reduction current obtained through an electron carrieror the quantity of the electric current may be measured. Examples areshown in “Biosensor and Quantitative Assay of Substrate Using the Same(Application No. JP 09-263492)” and the like.

In addition, for example, daily monitoring of urinary myo-inositollevels can be more easily carried out by incorporating the above sensordirectly into a toilet stool or the like or into a device attachedthereto. Such a device may further have functions of memorizing themeasurements and of connecting to a terminal of an informationprocessor. In this way, even when the subject stays in a distant place,a medical practitioner or medical institution can make contact with thesubject through an electric medium to manage vital data; to give amedical advice; and to examine the degree of impaired glucose toleranceand the degree of insulin secretory defect, leading to review of thediet contents, control of the amount of exercise, improvement of lifestyle, and the medical treatment.

In the case of making quantitative determination of glucose togetherwith myo-inositol in a sample to detect mild impaired glucose toleranceand/or insulin secretory defect, myo-inositol is quantitativelydetermined preferably by the method of the present invention, whileglucose may be quantitatively determined using any conventional methods.

In addition, more precise management can be performed by combining theresults of the determination and a doctor's observation. Furthermore,because it is possible to determine the risk to develop diabetes byfinding the precondition to diabetes, i.e. prediabetes, and also mildimpaired glucose tolerance and insulin secretory defect, which are notprediabetic at present but are highly likely to change to diabetes orprediabetes in the near future, though such finding being impossible bya conventional marker, for example, the risk can be used as an item ofexamination for life insurance or the like.

For quantitatively determining myo-inositol in a sample, 1 to 500 μL ofthe sample is added to the composition for myo-inositol quantitativedetermination to allow a reaction at 37° C. and then the amounts of acoenzyme changed may be directly or indirectly determined for severalminutes or several tens of minutes between two time points after thereaction starts, for example, for 1 minute between 3 minutes and 4minutes after the reaction initiation or for 5 minutes between 3 minutesand 8 minutes. In this case, the myoinositol content in the sample canbe determined by making a comparison with changes in absorbance whichare measured for known concentrations of myo-inositol.

The composition (reagent) for the quantitative determination need tocontain at least an enzyme that acts on myo-inositol and preferably itfurther contains a coenzyme.

In addition, a surfactant such as polyoxyethylene octylphenyl ether(OP-10) may be added to the present reagent as appropriate.

Furthermore, the present reagent is used in a form of a liquid product,a freeze-dried product, or a frozen product.

For quantitatively determining myo-inositol in a sample, any types ofmethods using an enzyme to quantitatively determine myo-inositol may beused. The enzyme to be used in the present invention, which is capableof quantitatively determining myo-inositol, includes any enzymes thatact on at least myo-inositol. Of those, however, myo-inositoldehydrogenase is preferable, and myo-inositol dehydrogenase derived fromFlavobaterium sp. 671 (FERM BP-7323, hereinafter abbreviated asF.sp.671) is most preferable. In addition, preferably, the myo-inositoldehydrogenase to be used has as low as possible or no contamination ofsubstances that adversely affects coenzymes such as thio-NAD and NADH inthe reagent, for example, substances having the activity of decomposingcoenzymes, such as NADH oxidase.

The strain F.sp.671 is deposited on an international basis with thedeposit number of FERM BP-7323 (date of deposit: Oct. 12, 2000) at theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, The Ministry of International Tradeand Industry, located at 1-1-3 Higashi, Tsukuba, Ibaraki, Japan (atpresent: International Patent Organism Depositary, the NationalInstitute of Advanced Industrial Science and Technology, IndependentAdministrative Agency, located at Center 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan).

For the detection of myo-inositol, any types of methods capable ofdetecting myo-inositol may be used. The methods include: a method usinga visible light coloring reagent, for example, typically yellow coloringwith thio-NAD, blue coloring with nitro blue tetrazolium (NBT), or redcoloring with 2-(4iodophenyl)3-(4nitrophenyl)-5-phenyl-2H-tetrazoliumchloride (INT); a luminescence method; a fluorescence method; a methodinvolving detection of an electric change; and a combination of thesemethods with amplification techniques.

In addition, using a compact device which can utilize any one of theabove methods, the determination of urinary myo-inositol can be carriedout non-invasively without restriction on time and place.

The assay for determining the activity of myo-inositol dehydrogenase isas follows:

(1) Activity Assay

<Composition of Reaction Solution>

100 mM Tris buffer (pH 8.5)

20 mM myo-inositol (Sigma Co., Ltd.)

2 mM nicotinamide adenine dinucleotide (NAD) (Oriental Yeast Co., Ltd.)

5 U/ml diaphorase (Asahi Kasei Corporation)

0.025% nitro blue tetrazolium (NBT; Wako Pure Chemical Industries, Ltd.)

1.5% Triton-X100 (Wako Pure Chemical Industries, Ltd.)

One ml of the above reaction solution is added to a small test tube.After the reaction solution is incubated at 37° C. for 5 minutes, 20 μlof an enzyme solution diluted by B times is added thereto and mixed tostart the reaction. After the reaction exactly for 5 minutes, 2 ml of0.1 N HCl is added and mixed to stop the reaction. An absorbance at 550nm is measured to obtain A1. In addition, the same reaction solutionexcluding myo-inositol is used to carry out a similar measurement toobtain the absorbance A0. The enzyme activity can be calculated from thefollowing equation.U/ml=[(A1−A0)/18.3]×[1/5]×[3.02/0.02]×B

Numerals in the equation represent the following meanings.

18.3: Molar absorption coefficient of NTB

5: Reaction time

3.02: Total volume of reaction solution

0.02: Volume of enzyme solution

B: Dilution factor of enzyme solution

The properties of myo-inositol dehydrogenase derived from the strainF.sp.671 are as follows:

(2) Enzyme Action

This enzyme produces inosose and a reduced coenzyme in the presence ofat least myo-inositol and a coenzyme. The coenzyme includes nicotinamideadenine dinucleotides (hereinafter abbreviated as NADs) such asnicotinamide adenine dinucleotide (NAD), acetylpyridine adeninedinucleotide (acetyl-NAD), nicotinamide hypoxanthine dinucleotide(deamino-NAD), pyridine aldehyde adenine dinucleotide (aldehyde-NAD),nicotinamide adenine dinucleotide phosphate (NADP), thio-nicotinamideadenine dinucleotide (thio-NAD), and thio-nicotinamide adeninedinucleotide phosphate (thio-NADP).

Table 1 shows the ratio of relative activities on use of each coenzyme(as 100% when NAD is used as a coenzyme). The relative activities weredetermined with the coenzyme changed according to the following method.

Relative Activity Assay

<Composition of Reaction Solution>

Buffer: 100 mM glycine buffer (pH 10.0)

Substrate: 20 mM myo-inositol (Sigma, Co., Ltd.)

Coenzyme: 2 mM

-   -   (NAD, thio-NAD, NADP, thio NADP; Oriental Yeast Co., Ltd.)

One ml of the above reaction solution is added to a quartz cell. Then,the quartz cell is placed in a spectrophotometer adjusted at atemperature of 37° C. The cell is incubated for 5 minutes or more andthen 20 μl of an enzyme solution of about 1.0 U/ml is added thereto andmixed. The initial velocity is obtained from an absorbance change perminute at a wavelength peculiar to each reduced coenzyme. The initialvelocity obtained with each coenzyme is compared with the initialvelocity (100%) obtained using NAD as a coenzyme to provide the relativeactivity. TABLE 1 Relative activity ratio for each coenzyme used Name ofbacterial strain F.sp.671 Coenzyme myo-Inositol NAD 100% NADP 8%Thio-NAD 29% Thio-NADP 0%

(3) Substrate Specificity

According to the relative activity assay described above, themeasurement was performed using the same concentration ofD-chiro-inositol, D-mannose, D-fructose, D-galactose, mannitol,epi-inositol, or scyllo-inositol in place of the substrate in thereaction solution. Table 2 shows the enzyme activity for each substratereferring to the initial velocity of the reaction to myo-inositol as100%. It is revealed that the enzyme derived from the strain F.sp.671 isdehydrogenase having high specificity to myo-inositol.

The substrates used include D-mannose, D-fructose, D-galactose,mannitol, D-chiro-inositol (as above: Wako Pure Chemical Industries,Ltd.), myo-inositol, epi-inositol, and scyllo-inositol (as above: Sigma,Co., Ltd.). TABLE 2 Substrate specificity Name of bacterial strainF.sp.671 Coenzyme NAD myo-Inositol 100% chiro-Inositol  18%scyllo-Inositol less than 1% epi-Inositol  2% Galactose less than 1%Fructose less than 1% Mannose less than 1% Mannitol  0%

(4) Optimum pH

Following the relative activity assay described above, the measurementwas performed using each of 100 mM tris buffer (pH 7.0-9.0) and 100 mMglycine buffer (pH 9.0-11.0) in place of 100 mM of pH 10.0 glycinebuffer in the reaction solution. The measurement showed that the optimumpH was about 11.0 (substrate: myo-inositol).

(5) Molecular Weight

Used were TSK gel G300SW (0.75 Φ×600 mm), eluent: 50 mM phosphate buffer(pH 7.5) +0.2 M Na₂SO₄+0.05% NaN₃, and a molecular marker set ofOriental Yeast Co., Ltd. (Japan), a chromatography apparatus made byShimadzu Corporation (Japan). For the detection, the absorbance at UV280 nm and the activity of each fraction were measured. myo-Inositol wasused as a substrate in the activity measurement, revealing the molecularweight of 40,000±10,000.

(6) Heat Stability

The enzyme showed almost 100% remaining activity after treatment at 40°C. for 15 minutes. The enzyme solution of about 5 U/ml was subjected toheat treatment for 15 minutes. The remaining activity was measured usingthe enzyme activity assay described above. In the activity measurement,myo-inositol was used as a substrate.

(7) Km value

Using the relative activity assay described above, the concentration ofmyo-inositol and the concentrations of NAD and thio-NAD were changed todetermine Km values respectively. Using the activity assay describedabove, the substrate concentration was changed to calculate the Kmvalue.

Km value for substrate

-   -   myo-Inositol: 1.7±0.2 mM

Km value for coenzyme

-   -   NAD: 0.04±0.01 mM    -   Thio NAD: 4.5±1 mM

For the quantitative determination of myo-inositol with highersensitivity, the enzymatic cycling method can be used. An example of theenzymatic cycling method is illustrated in the following equation.

In the equation, A1 represents NAD(P) or thio-NAD(P); A2 represents areduced form of A1; B1 represents reduced NAD(P) when A1 is thio-NAD(P)or reduced thio-NADP) when A1 is NAD(P); and B2 represents an oxidizedproduct of B1. As used herein, NAD(P) represents nicotinamide adeninedinucleotide and nicotinamide adenine dinucleotide phosphate.

For the solution composition of the quantitative reaction ofmyo-inositol using the enzymatic cycling, two or more of coenzymes areappropriately selected in view of Km values of respective coenzymes ofmyo-inositol dehydrogenase, and the like, and subsequently the pHcondition is adjusted between the optimal pH values of forwardreaction/reverse reaction to make an efficient progress in the enzymaticcycling. The amounts of A1 and B 1 should be excess over themyo-inositol content in a sample and also excess over Km values ofmyo-inositol dehydrogenase for A1 and B1.

When using, for instance, myo-inositol dehydrogenase derived fromF.sp.671, the Km values for NAD and thio-NAD are 0.04 mM and 4.5 mM,respectively. For the cycling reaction, thio-NAD and NADH may beselected as coenzymes. The concentrations of A1 and B1 are preferably0.02 mM to 2 M, particularly preferably 0.05 to 100 mM. The amount ofmyo-inositol dehydrogenase is preferably 1 to 1000 U/mL, particularlypreferably 1 to 100 U/mL. The amounts can be suitably selected on thebasis of type and amount of the test sample, the myo-inositol content inthe sample to be assayed, and the like; but other amounts may be alsoallowed.

When hexokinase is used as an enzyme for eliminating sugars presented ina sample, any hexokinase capable of catalyzing the reaction from glucoseto glucose-6-phosphate may be used, including hexokinase derived fromBacillus sp. Preferable hexokinase is one having excellent heatstability. The hexokinase having excellent heat stability can beobtained by the method described in “Stable Hexokinase and ProductionMethod Thereof” (JP 2000-078982 A).

Because ADP generated together with glucose-6-phosphate has someinhibitory effect on the reaction in the enzymatic cycling method, thepresent inventors successfully have used ADP-dependent hexokinasesimultaneously with hexokinae to improve substantially the eliminationof glucose without any influence on the reaction of myo-inositoldehydrogenase.

Glc+ATP+Mg²⁺→G-6-P+ADP

Glc+ADP+Mg²⁺→G-6-P+AMP

ATP: Adenosine-5′-triphosphate

ADP: Adenosine-5′-diphosphate

AMP: Adenosine-5′-monophosphate

The assay of hexokinase activity is conducted as follows.

<Composition of Reaction Solution>

50 mM Tris buffer (pH 8.5) (Sigma, Co., Ltd.)

20 mM glucose (Wako Pure Chemical Industries, Ltd.)

4 mM ATP (Oriental Yeast Co., Ltd.)

5 U/mL glucose-6-phosphate dehydrogenase (Toyobo Co., Ltd.)

1 mM NADP (Oriental Yeast Co., Ltd.)

10 mM magnesium chloride (Wako Pure Chemical Industries, Ltd.)

Solution for dissolving and diluting the enzyme: 50 mM Tris buffer (pH8.5)

One mL of the above reaction solution is added to a quartz cell with1-cm optical path length, and incubated at 37° C. for 5 minutes. Then,20 μL of the enzyme solution, which is diluted B times, is added theretoand mixed to start the reaction. The absorbance at 340 nm is measuredfrom the initiation of the reaction to obtain the absorbance change A1per minute, which shows a linear reaction. A blind test is alsoconducted in a similar reaction to obtain the absorbance change A0 perminute, except that 50 μL of the solution for dissolving and dilutingthe enzyme is added instead of the enzyme solution. The enzyme activityis calculated from the following equation.U/ml =[(A1−A0)/6.22]×[1.02/0.02]×B

Numerals in the equation represent the following meanings.

6.22: Millimolar extinction coefficient of NADPH at 340 nm

1.02: Total volume of reaction solution (mL)

0.02: Volume of enzyme solution used in the reaction (mL)

B: Dilution factor of enzyme solution

The assay of ADP-dependent hexokinase activity is conducted as follows.

<Composition of Reaction Solution>

50 mM Tris buffer (pH 7.5)

20 mM glucose solution (Wako Pure Chemical Industries, Ltd)

2 mM ADP solution (pH 7.0) (Oriental Yeast Co., Ltd.)

5 U/mL glucose-6-phosphate dehydrogenase (Asahi Kasei Corporation)

1 mM NADP solution (Oriental Yeast Co., Ltd)

2 mM magnesium chloride solution (Wako Pure Chemical Industries, Ltd)

Solution for dissolving and diluting the enzyme: 10 mM Tris buffer (pH7.5)

Three mL of the above reaction solution is added to a small test tube,and incubated at 37° C. for 5 minutes. Then, 50 μL of the enzymesolution, which is diluted by B times, is added thereto and mixed tostart the reaction. The absorbance at 340 nm is measured from theinitiation of the reaction to obtain the absorbance change A1 perminute, which shows a linear reaction. A blind test is also conducted ina similar reaction to obtain the absorbance change A0 per minute, exceptthat 50 μL of the solution for dissolving and diluting the enzyme isadded instead of the enzyme solution. The enzyme activity is calculatedfrom the following equation.U/ml =[(A1−A0)/6.22]×[3.05/0.05]×B

Numerals in the equation represent the following meanings.

6.22: Millimolar extinction coefficient of NADPH at 340 nm

3.05: Total volume of reaction solution (mL)

0.05: Volume of enzyme solution used in the reaction (mL)

B: Dilution factor of enzyme solution

The amount of hexokinase is preferably 1 to 1,000 u/mL, particularlypreferably 1 to 100 u/mL. The amount of ADP-dependent hexokinase ispreferably 1 to 1,000 u/ml, particularly preferably 1 to 100 u/mL. Theamounts can be appropriately selected depending on type and amount ofthe test sample, and other amounts can be also used.

In addition, for the determination of urinary myo-inositol over a widerange of its concentration with good reproducibility, an enzymaticcycling reaction should be effectively performed. As a result ofintensive examination on concentrations and ratio of thio-NAD and NADH,two coenzymes to be used in the enzymatic cycling reaction, the presentinventors have found that the thio-NAD level is preferably 0.01 mM ormore, particularly preferably 2 to 10 mM in a final concentration andthe ratio of NADH/thio-NAD is preferably 0.01 to 0.5, particularlypreferably 0.01 to 0.1. However, the amounts can be appropriatelyselected according to type and amount of the test sample, and otheramounts may be applied.

EXAMPLES

The examples of the present invention and reference examples will bedescribed in detail, but the present invention is not limited thereto.

Reference Example 1 Study on thio-NAD Levels

1) Reagents

<R-1>

-   -   5 mM MES (2-Morpholinoethanesulfonic acid) (pH 6.0)    -   0 to 40 mM thio-NAD (Oriental Yeast Co., Ltd.)

<R-2; Reagent for myo-Inositol Quantitative Determination>

-   -   200 mM Bicine (pH 9.0)    -   0.3 mM NADH (Oriental Yeast Co., Ltd.)    -   25 u/mL myo-inositol dehydrogenase (Asahi Kasei Corporation)

2) Method

The measurement device used was Autoanalyzer 7170S (Hitachi ChemicalCo., Ltd.). To 3 μL of myo-inositol solution at concentrations of 0 to3,000 μM, 180 μL of R-1 reagent was added and incubated at 37° C. for4.8 minutes, followed by the addition of 180 μL of R-2 reagent to startthe reaction. Absorbances at 405 nm were measured at 5.4 and 7.8 minutesafter the reaction initiation, and then the difference therebetween wasobtained. An increasing rate of absorbance per minute (ΔmABS/min) wascalculated and the sensitivity was then investigated with respect to thestandard solution.

3) Results

The results are shown in FIG. 1. As shown in FIG. 1, using thio-NAD atfinal concentrations of 0.1 to 10 mM, the linearity of the calibrationcurve was observed over myo-inositol concentrations of 0 to 3,000 μm. Inaddition, it has been found that the final concentration of thio-NAD ispreferably 2 to 10 mM to enhance the sensitivity of myo-inositoldetection.

Reference Example 2 Study on Buffers in Reagents for myo-InositolQuantitative Determination

1) Reagents

<R-1>

-   -   5 mM MES (pH 6.0)    -   5 mM thio-NAD (Oriental Yeast Co., Ltd.)

<R-2; Reagent for myo-Inositol Quantitative Determination>

-   -   100 mM Buffer (pH 8.8)    -   0.5 mM NADH (Oriental Yeast Co., Ltd)    -   10 u/mL myo-inositol dehydrogenase (Asahi Kasei Corporation)

2) Method

R-2 reagents for myo-inositol quantitative determination were preparedfollowing the above shown composition with a buffer being selected from:

Tris (Tris(hydroxymethyl)aminomethane),

Tricine (N-Tris(hydroxymethyl)methylglycine),

Bicine (N,N-Bis(hydroxyethyl)glycine),

TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid),

TEA (Triethanolamine),

CHES (2-Cyclohexylamino)ethanesulfonic acid), and

AMPSO(3-((1,1-Dimethyl-2-hydroxy-ethyl)amino)-2-hydroxypropanesulfonicacid).

The measurement device used was Autoanalyzer 7170S (Hitachi ChemicalCo., Ltd.). To 15 μL of the standard 100-μM myo-inositol solutionprepared in advance, 180 μL of R-1 reagent was added and incubated at37° C. for 4.8 minutes, followed by the addition of 60 μL of R-2 reagentto start the reaction. Absorbances at 405 nm were measured at 5.4 and7.8 minutes after the reaction initiation, and then the differencetherebetween was obtained. An increasing rate of absorbance per minute(ΔmABS/min) was calculated and the sensitivity was then investigatedwith respect to the standard solution. The stability of each R-2 reagentwas investigated by an acceleration test wherein only R-2 reagent wasstored in an incubator at 30° C. for 20 days and then the same test asdescribed above was conducted on the 7th, 12th, and 20th days.

3) Results

The results are shown in FIG. 2. The buffers showing stable sensitivityin comparison to the standard solution were Tris, Tricine, Bicine andTEA; and the buffer having the most stable sensitivity was Bicine.

Reference Example 3 Study on ADP-Hexokinase

1) Reagents

-   -   <R-1>    -   5 mM MES (pH 6.0)    -   5 mM MgCl₂ (Wako Pure Chemical Industries, Ltd.)    -   8 mM ATP (Oriental Yeast Co., Ltd.)    -   10 mM thio-NAD (Oriental Yeast Co., Ltd.)    -   10 u/mL ATP-hexolinase (Asahi Kasei Corporation)    -   0 to 4 u/mL ADP-hexokinase (Asahi Kasei Corporation)

<R-2; Reagent for myo-Inositol Quantitative Determination>

-   -   200 mM Bicine (pH 9.0)    -   0.3 mM NADH (Oriental Yeast Co., Ltd.)    -   25 u/mL myo-inositol dehydrogenase (Asahi Kasei Corporation)

2) Method

The measurement device used was Autoanalyzer 7170S (Hitachi ChemicalCo., Ltd.). Samples were prepared by mixing 100 μL of 2,000 μMmyo-inositol solution and 1 mL of 0 to 10 g/dL glucose solution. To 3 μLof each sample, 180 μL of R-1 reagent was added and incubated at 37° C.for 4.8 minutes, followed by the addition of 180 μL of R-2 reagent tostart the reaction. Absorbances at 405 nm were measured at 5.4 and 7.8minutes after the reaction initiation, and then the differencetherebetween was obtained. An increasing rate of absorbance per minute(ΔmABS/min) was calculated and the sensitivity was then investigated forrespective samples.

3) Results

The results are shown in FIG. 3. As is evident from FIG. 3, when usingATP-hexolinase alone, the increasing glucose level, e.g. of 10 g/dLcauses an influence on the sensitivity. In contrast, when usingATP-hexolinase together with ADP-hexolinase, the increasing glucoselevel has no influence on the sensitivity. This indicates that glucosein a sample can be eliminated by simultaneous reaction withATP-hexolinase and ADP-hexokinase; and thus myo-inositol level can bedetermined more accurately.

Reference Example 4 myo-Inositol Quantitative Determination with HighSensitivity using Enzyme

1) Reagents

Reagent for myo-Inositol Assay

<R-1; Glucose Eliminating Reagent>

-   -   5 mM MES (pH 6.0)    -   0.05% NaN₃ (Wako Pure Chemical Industries, Ltd.)    -   0.05% OP-10 (Nippon Chemicals)    -   5 mM MgCl₂ (Wako Pure Chemical Industries, Ltd.)    -   8 mM ATP (Oriental Yeast Co., Ltd.)    -   10 mM thio-NAD (Oriental Yeast Co., Ltd.)    -   10 u/mL ATP-hexolinase (Asahi Kasei Corporation)    -   4 u/mL ADP-hexokinase (Asahi Kasei Corporation)

<R-2; Reagent for myo-Inositol Quantitative Determination>

-   -   200 mM Bicine (pH 9.0)    -   0.05% NaN₃ (Wako Pure Chemical Industries, Ltd.)    -   40 mM KHCO₃ (Wako Pure Chemical Industries, Ltd.)    -   0.3 mM NADH (Oriental Yeast Co., Ltd.)    -   25 u/mL myo-inositol dehydrogenase (Asahi Kasei Corporation)

2) Method

The measurement device used was Autoanalyzer 7170S (Hitachi ChemicalCo., Ltd.). To 3 μL of the myo-inositol solution prepared in advance,180 μL of the glucose eliminating reagent was added and incubated at 37°C. for 4.8 minutes for the glucose eliminating reaction, and then 180 μLof the reagent for myo-inositol quantitative determination was addedthereto to start the reaction. Absorbances at 405 nm were measured at5.4 and 7.8 minutes after the reaction initiation, and then thedifference therebetween was obtained. An increasing rate of absorbanceper minute (ΔmABS/min) was calculated.

3) Results

The results are shown in FIG. 4. As shown in FIG. 4, the present assayreagent allowed quantitative determination of myo-inositol in a simpleway. The measurement range of myo-inositol was 0 to 2,000 μM and thelower limit of detection was 10 μM when the lower limit of detection wasdefined as the minimum concentration, which does not overlap “the meanof ΔmABS/min+2×standard deviation” obtained by multiple measurements of0 mM myo-inositol.

Example 1 Detection of Mild Impaired Glucose Tolerance by Determinationof Urinary myo-Inositol

1) Subjects:

One hundred and twelve subjects were examined by the standard 75 g oralglucose load test. Blood samples were collected just before the glucoseload, and at 30, 60, 120, and 180 minutes after the glucose load todetermine levels of blood glucose and insulin. Simultaneously, urinesamples were collected just before the glucose load, and at 60, 120, and180 minutes after the glucose load to determine levels of myo-inositol,urinary glucose, and creatinine.

2) Reagents and Assays:

Blood glucose: Electrode method (Kyoto Daiichi Kagaku Corporation:GA-1160)

Insulin: RIA2 Antibody method

myo-Inositol Assay Reagent: the same as that of Example 4

Urinary glucose: Electrode method (Kyoto Daiichi Kagaku Corporation:GA-1160)

Creatinine: Creatinine-HA Test Wako (Wako Pure Chemical Industries,Ltd.)

3) Method:

ΣPG, the total of blood glucose levels just before 75 g oral glucoseload, and at 30, 60, and 120 minutes after the glucose load, was used asan index of glucose tolerance. myo-Inositol level and creatinine levelin respective urine samples just before the 75 g oral glucose load, andat 30, 60, and 120 minutes after the glucose load were determined tocalculate the myo-inositol amount to the amount of urinary creatinineexcreted (myo-inositol/creatine). In addition, Δ myo-inositol content[(myo-inositol content at 60 min−myo-inositol content beforeload)/2]+[(myo-inositol content at 120 min−myo-inositol content beforeload)/2] was used as an index of myo-inositol level between before andafter the glucose load The relationship between ΣPG and Δ myo-inositolcontent was investigated.

4) Results:

The results are shown in Table 3 and FIG. 5. As shown in FIG. 5, ΣPG andΔ myo-inositol content showed a very good correlation. Higher ΣPGindicates that blood glucose levels are kept higher after the glucoseload, and thus the presence of impaired glucose tolerance. In addition,for example, if the characteristic value of Δ myo-inositol is set as 10μg/mg Cre (creatinine), an effective detection can be made for caseshaving mild impaired glucose tolerance with ΣPG level of about 530mg/dL. Blood glucose (mg/dL) Insulin (μU/mL) Urinary myo-inositol (μg/mgCre) Urinary Glucose (g/dL) Δmyo- 0 min. 30 60 120 180 ΣPG 0 min. 30 60120 180 I.I 0 min. 60 120 180 Δmyo 0 min. 60 120 180 Inositol ΣPGΔ|R|/ΔPG 1 Normal 89 150 161 135 94 535 5 23 23 22 13 0.30 13 24 33 2615 0.03 0.02 0.02 0.01 15 535 0.30 2 Normal 105 160 153 65 107 483 6 5940 24 22 0.96 23 24 40 26 9 0.02 0.01 0.01 0.01 9 483 0.96 3 Normal 102119 158 121 76 500 2 26 47 20 11 1.41 11 12 13 13 1 0.01 0.02 0.02 0.021 500 1.41 4 Normal 85 170 143 98 85 496 5 41 76 21 14 0.42 46 60 51 439 0 0.07 0.01 0.01 9 496 0.42 5 Normal 106 164 162 115 83 547 5 17 19 208 0.21 13 13 19 15 3 0.02 0.03 0.03 0.02 3 547 0.21 6 Normal 96 125 139134 125 494 5 10 24 25 16 0.17 26 38 69 68 23 0.01 0.01 0.01 0.01 23 4940.17 7 Normal 92 130 139 90 78 451 5 16 13 19 11 0.29 21 24 32 29 7 0.010.02 0.02 0.01 7 451 0.29 8 Normal 101 144 135 108 107 488 7 37 27 36 340.70 24 17 23 22 −4 0.02 0.03 0.03 0.03 −4 488 0.70 9 Normal 87 161 172109 60 529 3 25 21 17 4 0.30 25 35 46 44 16 0.01 0.03 0.01 0.01 16 5290.30 10 Normal 106 175 167 133 60 581 13 83 124 107 14 1.01 17 26 35 2514 0.02 0.01 0 0 14 581 1.01 11 Normal 106 158 129 128 72 521 12 63 5073 11 0.98 9 16 14 14 6 0.01 0 0.01 0 6 521 0.98 12 Normal 101 137 10183 82 422 11 87 86 25 10 2.11 9 12 20 13 7 0.01 0.01 0 0 7 422 2.11 13Normal 98 129 81 101 97 409 7 49 10 18 11 1.35 13 23 18 20 8 0.01 0 00.01 8 409 1.35 14 Normal 92 127 123 85 70 427 9 39 25 18 8 0.86 11 1211 12 0 0.02 0.01 0.01 0 0 427 0.86 15 Normal 95 132 103 102 69 432 8 3930 28 12 0.84 9 12 11 11 3 0.01 0.01 0.01 0.01 3 432 0.84 16 Normal 106167 107 102 85 482 10 75 32 21 9 1.07 9 11 12 12 2 0.03 0.03 0.02 0.02 2482 1.07 17 Normal 88 121 138 100 79 447 6 18 32 15 5 0.36 9 10 15 17 40.01 0.01 0.01 0.01 4 447 0.36 18 Normal 97 130 88 103 102 418 5 28 1814 8 0.70 14 13 11 11 −2 0.01 0.01 0.01 0.01 −2 418 0.70 19 Normal 99151 114 105 106 469 7 7 8 24 16 0.00 8 10 12 18 3 0.02 0.02 0.02 0 3 4690.00 20 Normal 102 148 178 127 89 553 8 62 59 38 20 1.17 41 70 92 47 400.01 0.24 0.4 0.03 40 553 1.17 21 Normal 89 108 120 92 102 409 10 51 6521 37 2.16 35 34 40 31 2 0.01 0.01 0.01 0.01 2 409 2.16 22 Normal 91 144153 105 72 493 9 75 67 38 7 1.25 28 26 25 17 −2 0.01 0.03 0.02 0.01 −2493 1.25 23 Normal 101 154 148 91 73 494 5 26 36 14 5 0.40 7 8 8 9 10.02 0.02 0.02 0.01 1 494 0.40 24 Normal 96 148 177 124 52 545 10 29 5967 10 0.37 11 11 11 11 0 0.02 0.03 0.02 0 0 545 0.37 25 Normal 95 168 9884 76 445 6 78 20 16 4 0.99 24 34 30 31 8 0.01 0.02 0.01 0 8 445 0.99 26Normal 89 147 93 84 55 413 5 26 23 21 4 0.36 7 9 14 8 5 0.02 0.01 0 0.015 413 0.38 27 Normal 90 129 93 101 81 413 7 36 14 17 6 0.74 25 22 20 19−4 0 0.01 0.01 0 −4 413 0.74 28 Normal 75 96 75 84 60 330 3 37 17 13 41.62 11 13 13 14 2 0.03 0.03 0.02 0.02 2 330 1.62 29 Normal 92 127 87 8369 389 5 37 25 11 5 0.91 25 21 20 18 −4 0.03 0.01 0 0.01 −4 389 0.91 30Normal 83 109 107 101 78 400 9 32 27 26 12 0.88 16 15 17 14 0 0 0.01 0 00 400 0.88 31 Normal 100 123 71 89 79 383 7 32 33 13 3 1.09 5 8 13 10 50.03 0 0 0 5 383 1.09 32 Normal 96 110 75 89 82 370 6 46 20 18 7 2.86 1518 17 12 2 0.01 0.01 0 0.02 2 370 2.86 33 Normal 91 108 82 80 82 361 716 17 19 8 0.53 10 13 16 15 4 0.02 0.01 0 0 4 361 0.53 34 Normal 91 140152 99 59 482 3 2 5 7 3 −0.02 7 8 8 7 1 0.01 0.01 0.01 0 1 482 −0.02 35Normal 79 119 120 89 60 407 4 9 5 7 2 0.13 10 12 15 14 4 0.02 0.01 0.010.01 4 407 0.13 36 Normal 100 151 148 139 100 538 10 41 45 49 27 0.61 1826 38 36 14 0.02 0.03 0.03 0 14 538 0.61 37 Normal 96 119 110 85 63 4105 38 34 18 5 1.43 20 22 28 29 5 0.01 0.01 0 0 5 410 1.43 38 Normal 99145 149 109 63 502 9 21 31 17 7 0.26 23 36 40 32 15 0.01 0.02 0.01 0 15502 0.26 39 Normal 85 98 101 82 50 366 4 14 4 0.77 8 8 8 9 0 0.01 0.010.02 0 0 386 0.77 40 Normal 89 114 87 91 63 381 7 9 10 15 3 0.08 27 3544 44 12 0.01 0.02 0.01 0 12 381 0.08 41 Normal 95 169 175 109 59 548 1135 46 29 7 0.32 9 10 9 9 0 0 0.02 0.01 0.01 0 548 0.32 42 Normal 76 7866 90 84 310 2 7 4 10 5 2.50 8 8 8 8 −1 0.01 0.01 0.02 0.01 −1 310 2.5043 Normal 100 124 92 87 65 403 8 51 29 18 5 1.79 8 7 9 12 1 0.01 0.030.01 0 1 403 1.79 44 Normal 95 138 117 91 67 441 7 26 44 27 6 0.44 10 1723 26 10 0.02 0 0 0 10 441 0.44 45 Normal 89 106 85 82 79 362 6 79 32 164 4.29 7 8 9 12 2 0.02 0.01 0.01 0 2 362 4.29 46 Normal 103 192 153 9680 544 9 33 44 20 5 0.27 18 56 38 28 28 0.02 0.77 0.06 0 28 544 0.27 47Normal 87 131 123 109 69 450 6 42 36 24 4 0.82 7 10 10 9 3 0.01 0.010.01 0.01 3 450 0.82 48 Normal 99 126 104 83 66 412 10 47 9 15 5 1.37 2718 21 18 −7 0 0.02 0 0.01 −7 412 1.37 49 Normal 88 106 86 85 89 363 3 2218 17 8 0.95 14 20 19 14 5 0 0 0 0 5 363 0.95 50 Normal 84 131 89 89 80393 6 40 24 12 10 0.72 10 15 17 16 5 0.03 0 0 0 5 393 0.72 51 Normal 85116 99 110 83 410 6 17 17 18 9 0.35 13 13 12 11 −1 0.03 0.02 0.02 0 −1410 0.35 52 Normal 87 122 89 96 88 394 6 27 30 35 13 0.60 11 16 20 24 70.02 0.02 0.01 0 7 394 0.60 53 Normal 90 147 112 116 52 465 6 72 53 50 81.16 17 16 22 26 2 0 0 0 0 2 465 1.16 54 Normal 92 187 186 133 49 598 317 25 27 4 0.15 368 487 623 395 186 0 0.23 0.15 0.03 186 598 0.15 55Normal 99 146 181 133 137 559 5 19 16 15 13 0.30 4 4 4 3 1 0 0 0.01 0 1559 0.30 56 Normal 99 160 206 138 68 603 11 30 49 72 18 0.31 7 7 14 10 40.03 0.04 0 0 4 603 0.31 57 Normal 107 151 189 115 44 562 6 16 44 38 70.23 13 20 42 30 18 0.02 0.03 0.05 0.02 18 562 0.23 58 Normal 105 194234 110 81 643 5 17 40 23 11 0.13 24 32 72 29 28 0 0.04 0.13 0 28 8430.13 59 Normal 89 167 187 130 108 573 22 133 142 104 79 1.42 26 34 41 2712 0.02 0.02 0.01 0 12 573 1.42 60 IFG 117 165 154 90 80 526 8 37 108 2711 0.60 24 46 72 45 35 0.01 0.06 0.08 0 35 526 0.60 61 IFG 115 174 206136 65 631 4 8 19 20 4 0.07 12 33 84 25 46 0.01 0.23 0.55 0 46 631 0.0762 IFG 117 204 220 103 152 644 7 31 37 33 16 0.28 37 113 140 56 90 0.010.61 1.04 0.12 90 644 0.28 63 IFG 110 185 240 127 120 662 8 15 28 15 140.12 17 26 59 29 26 0.02 0.05 0.12 0.03 26 682 0.12 64 IFG 111 183 20083 75 577 4 16 22 12 4 0.17 16 26 48 28 22 0 0.1 0.17 0 22 577 0.17 65IFG 110 153 60 88 82 411 5 44 22 18 15 0.91 13 15 19 19 4 0.01 0.01 0.020.01 4 411 0.91 66 IFG 117 236 192 101 71 646 24 196 100 48 17 1.45 1219 19 13 7 0.02 0.03 0.02 0.01 7 646 1.45 67 IFG 111 166 167 124 70 56812 53 55 40 8 0.75 9 22 21 14 13 0.01 0.02 0.03 0 13 568 0.75 68 IFG 113204 229 115 58 661 5 20 47 44 12 0.16 49 111 96 42 54 0.01 0.89 0.530.03 54 661 0.16 69 IGT 111 192 237 161 63 701 10 18 26 48 9 0.10 22 67146 94 85 0.01 0.37 1.31 0.41 85 701 0.10 70 IGT 104 153 130 141 141 5287 28 24 17 16 0.43 15 22 29 28 10 0.02 0.02 0 0.01 10 528 0.43 71 IGT113 195 224 170 147 702 7 19 44 48 31 0.15 63 114 185 176 86 0.01 0.670.38 0.14 86 702 0.15 72 IGT 98 158 213 143 48 612 5 14 22 33 5 0.15 5972 149 75 52 0.03 0.19 1.16 0.17 52 612 0.15 73 IGT 95 119 147 142 73503 5 20 21 29 6 0.63 40 31 36 40 −6 0.01 0.01 0.03 0.01 −6 503 0.63 74IGT 103 187 217 164 111 671 6 18 30 35 11 0.14 21 55 74 27 44 0.01 0.020.01 0.01 44 671 0.14 75 IGT 123 196 232 150 158 701 3 30 60 67 56 0.3731 88 119 88 72 0.01 0.95 0.53 0.66 72 701 0.37 76 IGT 108 150 192 168124 618 3 17 22 32 26 0.33 15 14 15 13 0 0.02 0.02 0.02 0.02 0 618 0.3377 IGT 114 178 204 172 136 668 4 10 13 17 12 0.09 29 47 89 49 39 0.020.04 0.13 0.03 39 668 0.09 78 IGT 108 211 214 167 69 700 6 36 50 41 100.29 15 66 116 28 76 0.01 0.22 0.43 0.03 76 700 0.29 79 IGT 97 211 256196 117 760 6 20 28 34 16 0.12 15 29 29 26 15 0.03 0.06 0.05 0.01 15 7600.12 80 IGT 123 218 244 160 83 745 2 11 13 21 7 0.09 17 73 150 27 950.02 1.41 1.54 0.02 95 745 0.09 81 IGT 100 161 204 173 101 638 7 9 12 1412 0.03 15 15 21 18 3 0.02 0.02 0.03 0.02 3 638 0.03 82 IGT 110 219 240186 100 755 6 15 21 31 13 0.08 32 117 210 178 131 0.03 3.12 4.13 1.44131 755 0.08 83 Diabetic 127 206 250 178 125 761 8 21 25 37 21 0.16 1514 36 27 11 0.02 0.03 0.14 0.08 11 761 0.16 84 Diabetic 128 249 279 21489 870 6 22 25 31 15 0.13 50 119 221 144 120 0.02 1.6 1.33 0.31 120 8700.13 85 Diabetic 151 228 263 256 166 898 5 12 13 16 7 0.09 50 152 262208 157 0.03 0.85 2.33 1.02 157 898 0.09 86 Diabetic 170 278 326 322 1971096 5 7 8 11 9 0.02 64 146 232 225 125 0.01 1.33 3.46 2.75 125 10960.02 87 Diabetic 204 281 337 363 286 1185 12 15 19 26 28 0.04 21 63 145151 83 0.05 3.42 7.25 7.25 83 1185 0.04 88 Diabetic 112 209 262 237 91820 4 10 18 15 7 0.06 33 106 195 112 117 0.01 1.17 0.98 0.16 117 8200.06 89 Diabetic 129 232 288 225 122 874 4 11 15 10 7 0.07 14 71 170 80106 0.03 2.82 5.89 1.16 106 874 0.07 90 Diabetic 106 171 253 243 115 7734 9 20 32 14 0.08 27 44 135 79 63 0.02 0.99 5.11 1.96 63 773 0.08 91Diabetic 110 203 232 201 105 746 8 41 43 46 14 0.38 16 83 112 90 82 0.021.57 1.92 1.25 82 746 0.38 92 Diabetic 139 225 286 326 245 976 4 5 8 108 0.01 36 86 191 202 103 0.05 2.71 6.43 8.73 103 976 0.01 93 Diabetic118 232 285 232 114 865 7 15 20 31 15 0.07 16 50 155 62 87 0.02 1.090.73 0.11 87 865 0.07 94 Diabetic 132 220 329 318 223 999 30 49 80 10574 0.22 38 40 244 167 104 0.03 0.12 0.4 0.61 104 999 0.22 95 Diabetic102 178 250 250 122 780 2 9 13 36 16 0.09 18 26 148 91 69 0 0.08 3.311.16 69 780 0.09 96 Diabetic 173 239 352 311 201 1075 9 7 23 36 20 −0.0349 108 236 227 123 0.04 2.21 6.46 4.85 123 1075 −0.03 97 Diabetic 152236 224 264 263 876 6 17 14 12 11 0.13 40 62 146 167 64 0.01 0.15 0.180.11 64 876 0.13 98 Diabetic 166 252 306 274 205 998 3 11 17 20 10 0.0915 71 161 114 100 0.01 1.38 4.5 1.68 100 998 0.09 99 Diabetic 101 249373 444 342 1167 3 5 7 6 5 0.01 37 154 290 301 185 0.02 3.44 2.45 2.1185 1167 0.01 100 Diabetic 162 239 314 299 162 1004 7 11 33 31 12 0.0533 56 174 127 83 0.03 0.36 3.29 2.23 83 1004 0.05 101 Diabetic 141 208261 189 138 799 3 4 7 11 8 0.01 28 66 154 67 82 0.02 0.12 0.66 0.02 82799 0.01 102 Diabetic 136 236 262 225 209 859 7 14 18 20 10 0.07 73 160268 181 141 0.01 0.92 1.76 0.53 141 859 0.07 103 Diabetic 101 128 193206 138 628 6 15 25 58 46 0.33 13 19 46 28 20 0.02 0 0.02 0.02 20 6280.33 104 Diabetic 110 198 241 257 151 806 5 24 32 55 31 0.22 45 55 99118 32 0.01 0.15 1.3 1.61 32 806 0.22 105 Diabetic 152 253 346 291 1581042 10 19 44 16 23 0.09 99 183 371 280 178 0.11 3.02 3.32 3.2 178 10420.09 106 Diabetic 129 239 202 130 86 700 5 39 65 24 6 0.31 24 69 67 4044 0.03 1.76 0.59 0.12 44 700 0.31 107 Diabetic 112 200 250 283 246 8456 11 15 29 27 0.06 33 66 157 178 78 0.01 0.42 1.62 2.5 78 846 0.06 108Diabetic 111 198 251 254 171 814 5 17 20 32 21 0.14 17 43 180 167 940.02 0.48 0.99 0.84 94 814 0.14 109 Diabetic 137 198 258 232 141 825 511 30 21 8 0.10 27 105 177 138 114 0.08 3.88 6.04 3.78 114 825 0.10 110Diabetic 134 201 276 233 151 844 5 13 19 20 11 0.12 22 43 138 85 68 0.020.65 4.88 2.69 68 844 0.12 111 Diabetic 111 169 240 201 101 721 4 12 2828 10 0.14 27 63 132 90 70 0.01 0.43 1.44 0.1 70 721 0.14 112 Diabetic136 201 200 137 141 674 7 8 10 20 13 0.02 26 35 38 31 11 0.01 0.01 0 011 674 0.02

Example 2 Detection of Impaired Early insulin Secretion by Determinationof Urinary myo-Inositol 1) Subjects:

The same as those of Example 1.

2) Reagents and Assays:

The same as those of Example 1.

3) Method:

myo-Inositol levels and creatinine levels in each urine sample justbefore the 75 g oral glucose load, and at 60 and 120 minutes after theglucose load were determined, and then the myo-inositol amount to theamount of urinary creatinine excreted (myo-inositol/creatinine) wasobtained. In addition, Δ myo-inositol content [(myo-inositol content at60 min−myo-inositol content before load)/2]+[(myo-inositol content at120 min−myo-inositol content before load)/2] was used as the index ofmyo-inositol level between before and after the glucose load. Therelationship between the insulinogenic index (I.I) and Δ myo-inositolcontent was investigated.

4) Results:

The results are shown in FIG. 6. As shown in FIG. 6, the relationshipbetween the insulinogenic index and Δ myo-inositol content was found. Ina large percentage of cases where the Δ myo-inositol content was 15 μmgCre or more, the insulinogenic index showed less than 0.4. According tothe guideline of the Japan Diabetes Society, the insulinogenic index ofless than 0.4 can judge the presence of impaired early insulinsecretion. As is evident from FIG. 6, if the characteristic value of Δmyo-inositol is 15 μg/mg Cre, an effective detection can be made forcases showing the insulinogenic index of less than 0.4, or havingimpaired early insulin secretion.

Example 3 Detection of Mild Impaired Glucose Tolerance by Determinationof Urinary myo-Inositol and Urinary Glucose

1) Subjects:

The same as those of Example 1.

2) Reagents and Assays:

The same as those of Example 1.

3) Method.

myo-Inositol levels and creatinine levels in each urine sample justbefore the 75 g oral glucose load, and at 60 and 120 minutes after theglucose load were determined. Then, the myo-inositol content to theamount of urinary creatinine excreted (myo-inositol/creatinine) wasobtained. At the same time, urinary glucose levels were also obtained. Δmyo-inositol content [(myo-inositol content at 60 min−myo-inositolcontent before load)/2]+[(myo-inositol content at 120 min−myo-inositolcontent before load)/2] was used as the index of myo-inositol levelbetween before and after the glucose load.

4) Results The case showing Δ myo-inositol content of 10 μg/mg Cre ormore was referred to as plus (+), while the other case was referred toas minus (−). Likewise for the urinary glucose, the case showing anurinary glucose level of 50 mg/dL or more at 2 hours after glucose loadwas referred to as “+”, while the other case was referred to as “−”. ΣPGvalues calculated in Example 5 were used.

Out of 112 subjects tested, 52 subjects were in the group of Δmyo-inositol (−) and urinary glucose (−), 12 subjects were in the groupof Δ myo-inositol (+) and urinary glucose (−), and 48 subjects were inthe group of Δ myo-inositol (+) and urinary glucose (+). Nobodycorresponded to the group of Δ myo-inositol (−) and urinary glucose (+).The respective ΣPG values of the group of Δ myo-inositol (−) and urinaryglucose (−), the group of Δ myo-inositol (+) and urinary glucose (−),and the group of Δ myo-inositol (+) and urinary glucose (+) werecompared with one another.

The results are shown in FIG. 7. As shown in FIG. 7, the mean andstandard deviation of ΣPG values of the group of Δ myo-inositol (−) andurinary glucose (−) were 453 mg/dL and 76.6 mg/dL, respectively. Themean and standard deviation of ΣPG values of the group of Δ myo-inositol(+) and urinary glucose (−) were 556 mg/dL and 81.1 mg/dL, respectively.The mean and standard deviation of ΣPG values of the group of Δmyo-inositol (+) and urinary glucose (+) were 791 mg/dL and 164.8 mg/dL,respectively. Furthermore, as compared with ΣPG of the group of Δmyo-inositol (−) and urinary glucose (−), ΣPG of the group of Δmyo-inositol (+) and urinary glucose (−) was significantly higher, andalso ΣPG of the group of Δ myo-inositol (+) and urinary glucose (+) wassignificantly much higher. These show the degree of impaired glucosetolerance could be determined non-invasively by determining urinarymyo-inositol and urinary glucose in combination.

Example 4 Detection of Impaired Early Insulin Secretion by Determinationof Urinary myo-Inositol and Urinary Glucose

1) Subjects:

The same as those of Example 1.

2) Reagents and Assays:

The same as those of Example 1.

3) Method:

The same as that of Example 3.

4) Results:

The case showing Δ myo-inositol content of 10 μg/mg Cre or more wasreferred to as plus (+), while the other case was referred to as minus(−). Likewise for the urinary glucose, the case showing an urinaryglucose level of 50 mg/dL or more at 2 hours after glucose load wasreferred to as “+”, while the other case was referred to as “−”. Theinsulinogenic index values calculated in Example 2 were used.

The respective insulinogenic index values (ΔIRI 30-0/ΔPG 30-0) of thegroup of Δ myo-inositol (−) and urinary glucose (−), the group of Δmyo-inositol (+) and urinary glucose (−), and the group of Δmyo-inositol (+) and urinary glucose (+) were compared with one another.

The results are shown in FIG. 8. As shown in FIG. 8, the mean andstandard deviation of ΔIRI 30-0/ΔPG 30-0 of the group of Δ myo-inositol(−) and urinary glucose (−) were 1.32 and 0.79, respectively. The meanand standard deviation of ΔIRI 30-0/ΔPG 30-0 of the group of Δmyo-inositol (+) and urinary glucose (−) were 0.45 and 0.42,respectively. The mean and standard deviation of ΔIRI 30-0/ΔPG 30-0 ofthe group of Δ myo-inositol (+) and urinary glucose (+) were 0.16 and0.19, respectively. Furthermore, as compared with ΔIRI 30-0/ΔPG 30-0 ofthe group of Δ myo-inositol (−) and urinary glucose (−), ΔIRI 30-0/ΔPG30-0 of the group of Δ myo-inositol (+) and urinary glucose (−) wassignificantly lower, and also ΔIRI 30-0/ΔPG 30-0 of the group of Δmyo-inositol (+) and urinary glucose (+) was significantly much lower.These show that the degree of impaired early insulin secretion could bedetermined non-invasively by determining urinary myo-inositol andurinary glucose in combination.

Example 5 Detection of Mild Impaired Glucose Tolerance by Determinationof Urinary myo-Inositol

1) Subjects:

Out of those shown in Example 1, 59 subjects judged as NGT showing thefasting blood glucose level of less than 110 mg/dl and the two-hourpostload glucose level of less than 140 mg/dl.

2) Regents and Assays:

The same as those of Example 1.

3) Method:

Out of subjects judged as NGT, those having the one-hour postloadglucose level of 180 mg/dL or more or the two-hour postload glucoselevel of 120 mg/dL or more were referred to as B group or cases showingslightly decreased glucose tolerance (mild impaired glucose tolerance)and the others were referred to as A group. Fifty nine subjects of NGTincluded 45 subjects of A group and 14 subjects of B group. Themyo-inositol content to the amount of urinary creatinine excreted(myo-inositol/creatinine) in A or B group was obtained by determiningmyo-inositol levels and creatinine levels in each urine sample justbefore 75 g oral glucose load, and at 60 and 120 minutes after theglucose load.

In addition, Δ myo-inositol content [(myo-inositol content at 60min−myo-inositol content before load)/2]+[(myo-inositol content at 120min−myo-inositol content before load)/2] was used as the index ofmyo-inositol level between before and after the glucose load.

4) Results:

The results are shown in FIG. 9. As shown in FIG. 9, the mean of Δmyo-inositol content of A group was 3.9 μg/mg Cre and the mean of Δmyo-inositol content of B group was 25.8 μg/mg Cre. As compared with Agroup, B group (mild impaired glucose tolerance) with slightly decreasedglucose tolerance resulted in higher Δ myo-inositol content.

Example 6 Detection of Insulin Secretory Defect by Determination ofUrinary myo-Inositol

1) Subjects:

The same as those of Example 1.

2) Reagents and Assays:

The same as those of Example 1.

3) Method:

Out of subjects judged as NGT, those having the insulinogenic index ofless than 0.4 were referred to as B group and the others were referredto as A group. Fifty nine subjects of NGT included 37 subjects of Agroup and 22 subjects of B group. The myo-inositol content to the amountof urinary creatine excreted (myo-inositol/creatinine) in A or B groupwas obtained by determining myo-inositol levels and creatinine levels ineach urine sample just before 75 g oral glucose load, and at 60 and 120minutes after the glucose load.

In addition, Δ myo-inositol content [(myo-inositol content at 60min−myo-inositol content before load)/2]+[(myo-inositol content at 120min−myo-inositol content before load)/2] was used as the index ofmyo-inositol level between before and after the glucose load.

4) Results

The results are shown in FIG. 10. As shown in FIG. 10, the mean of Δmyo-inositol content of A group was 4.4 μg/mg Cre and the mean of Δmyo-inositol content of B group was 16.9 μg/mg Cre. As compared with Agroup, B group with impaired early insulin secretion resulted in higherΔ myo-inositol content

Example 7 Relationship between Urinary myo-Inositol in Glucose Load Testand Urinary myo-Inositol in Meal Load Test

1) Subjects:

Out of those shown in Example 1, 52 subjects who agreed with taking ameal load test, which included 22 subjects of NGT (C group), 14 subjectsof borderline type (B group), and 16 subjects of diabetes meritus (Dgroup). Blood samples were collected before the meal and at 120 minutesafter the meal, and then blood glucose level and insulin level weredetermined. In addition, urine samples were collected before the mealand at 120 minutes after the meal, and then myo-inositol level, urinaryglucose level, and creatinine level were determined. 2) Reagent andAssays:

The same as those of Example 1.

3) Method:

After Blood samples and urine samples were collected in fastingcondition, the subjects ingested a meal. The meal included retort-packedcooked foods (Wellness Menus (Nichirei Corporation) and packed boiledrice (Sato Foods Industries, Co., Ltd.)) containing 91.6 g carbohydrate,31.0 g protein, 13.9 g fat, and 1.1 g sodium with 662 kcal energy. At120 minutes after taking the meal, blood samples and urine samples werecollected. The myo-inositol content to the amount of urinary creatinineexcreted (myo-inositol/creatinine) was obtained by determiningmyo-inositol level and creatinine level in each urine sample. Inaddition, Δ myo-inositol content [(myo-inositol content at 120min−myo-inositol content before load)] was used as an index ofmyo-inositol content between before and after the meal.

4) Results:

FIG. 11 shows the relationship between Δ myo-inositol (X axis) in theglucose load test and Δ myo-inositol (Y axis) in the meal load test. Asshown in FIG. 11, Δ myo-inositol in the glucose load test and Δmyo-inositol in the meal load test showed a very good correlation(Y=1.044X−2.0, r=0.83, P<0.0001) and showed almost the same values.These results reveal that mild impaired glucose tolerance or insulinsecretory defect can be detected by determining unary myo-inositollevels before and after a meal, even if the glucose load test is notperformed.

Example 8 Relationship between Urinary myo-Inositol in Glucose Load Testand Urinary myo-Inositol in Meal Load Test

1) Subjects:

The same as those of Example 7.

2) Reagents and Assays:

The same as those of Example 1.

3) Method:

The same as that of Example 7.

4) Results

FIG. 12 shows the relationship between Δ myo-inositol in the glucoseload test and Δ myo-inositol in the meal load test for each group. Asshown in FIG. 12, Δ myo-inositol in the glucose load test and Δmyo-inositol in the meal load test were very well consistent with eachother for each group. These results reveal that mild impaired glucosetolerance and insulin secretory defect can be detected by determiningurinary myo-inositol levels before and after a meal, even if the glucoseload test is not conducted.

Example 9 Relationship between Urinary myo-Inositol in Meal Load Testand Mild Impaired Glucose Tolerance in Urinary Glucose NegativeIndividuals

1) Subjects:

Out of those shown in Example 7, 32 individuals who were negative forurinary glucose (less than 50 mg/dL) at 2 hours after the meal. 2)Reagents and Assays:

The same as those of Example 1.

3) Method:

The same as that of Example 7, except that individuals having Δmyo-inositol of 7 μg/mg Cre or more in the meal load test were referredto as (+) group, and the others were referred to as (−) group.

4) Results:

The results are shown in FIG. 13. As shown in FIG. 13, even inindividuals with urinary glucose negative at 2 hours after the meal,many of those of Δ myo-inositol (+) group showed higher ΣPG in theglucose load test, as compared with those of Δ myo-inositol (−) group.These results reveal that, even in individuals with urinary glucosenegative at 2 hours after a meal, the degree of impaired glucosetolerance can be determined non-invasively by determining urinarymyo-inositol levels before and after a meal.

INDUSTRIAL APPLICABILITY

As described above, the present invention provides a method of detectingmild impaired glucose tolerance and/or impaired early insulin secretionin a non-invasive and convenient manner with good reproducibility.

Reference to Deposited Biological Material

(1) (a) Name and address of the deposition organization, to which thepresent biological material has been deposited:

Name: International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology;

Address: Tsukuba Center 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan;

(b) Date of Deposition to Organization of (a):

-   -   Oct. 12, 2000 (original deposition date);

(c) Deposit Number for the Deposition given by Organization of (a):

-   -   FERM BP-7323.

1. A method of detecting mild impaired glucose tolerance, characterizedin that the method comprises: quantitatively determining myo-inositollevel in a sample; and evaluating a case where the level shows acharacteristic value or more as mild impaired glucose tolerance orinsulin secretory defect.
 2. The method according to claim 1, whereinthe quantitative determination of myo-inositol level in the sample iscarried out using an enzyme.
 3. The method according to claim 2, whereinthe enzyme is myo-inositol dehydrogenase.
 4. The method according toclaim 2 or 3, wherein the quantitative determination of the myo-inositollevel using the enzyme is carried out by an enzymatic cycling method. 5.The method according to any one of claims 1 to 4, characterized in thatthe myo-inositol level is quantitatively determined after elimination ofsugars other than myo-inositol in the sample.
 6. The method according toclaim 5, characterized in that two kinds of kinases are simultaneouslyused for the reaction of eliminating sugars other than myo-inositol inthe sample.
 7. The quantitative method according to claim 6,characterized in that said two kinds of kinases are ATP-hexokinase andADP-hexokinase.
 8. The quantitative method according to any one ofclaims 4 to 7, characterized in that thio-NAD is used as a coenzyme at afinal concentration of 0.1 mM or more in the reaction of quantitativelydetermining myo-inositol.
 9. The quantitative method according to anyone of claims 4 to 7, characterized in that thio-NAD is used as acoenzyme at a final concentration of 2 to 10 mM in the reaction ofquantitatively determining myo-inositol.
 10. The method according to anyone of claims 1 to 9, wherein the sample is obtained before and afterglucose load, or before and after a meal.
 11. The method according toclaim 10, wherein the sample is urine.
 12. The method according to anyone of claims 1 to 11, characterized in that the sample is urine and thecharacteristic value is 0 to 20 μg/mg creatinine when measured as anincreasing amount of myo-inositol excreted in the urine after 75 gglucose load.
 13. The method according to any one of claims 1 to 11,characterized in that the sample is urine and the characteristic valueis 8 to 12 μg/mg creatinine when measured as an increasing amount ofmyo-inositol excreted in the urine after 75 g glucose load.
 14. Themethod according to any one of claims 1 to 13, characterized in thatglucose level in the sample is quantitatively determined in addition tomyo-inositol level in the sample.
 15. A method of eliminating glucose ina sample, characterized in that two kinds of kinases are simultaneouslyused for the reaction of eliminating glucose in the sample.
 16. Themethod of eliminating glucose according to claim 15, characterized inthat said two kinds of kinases are ATP-hexokinase and an ADP eliminatingagent.
 17. The method of eliminating glucose according to claim 16,wherein the ADP eliminating agent is ADP-hexokinase.
 18. A method ofquantitatively determining myo-inositol level in a sample enzymaticallyusing myo-Inositol dehydrogenase in the presence of thio-NAD or NADH,characterized in that two kinds of kinases are used in combination. 19.The method according to claim 18, characterized in that said two kindsof kinases are ATP-hexokinase and an ADP eliminating agent.
 20. Themethod of eliminating glucose according to claim 19, wherein the ADPeliminating agent is ADP-hexokinase.
 21. A composition for quantitativedetermination of myo-inositol, characterized in that the composition atleast comprises: 1) thio-NAD; 2) NADH; 3) myo-inositol dehydrogenase;and 4) two kinds of kinases.
 22. The composition for quantitativedetermination of myo-inositol according to claim 21, characterized inthat said two kinds of kinases are ATP-hexokinase and an ADP eliminatingagent.
 23. The composition for quantitative determination ofmyo-inositol according to claim 22, wherein the ADP eliminating agent isADP-hexokinase.
 24. The composition for quantitative determination ofmyo-inositol according to any one of claims 21 to 23, characterized inthat the composition further comprises a buffer selected from: Bicine(N,N-Bis(hydroxyethyl)glycine), Tris (Tris(hydroxymethyl)aminomethane),TEA (Triethanolamine), and Tricine(N-Tris(hydroxymethyl)-methylglycine).
 25. The composition forquantitative determination of myo-inositol according to any one ofclaims 21 to 24, characterized in that the final concentration ofthio-NAD is 0.1 mM or more.
 26. The composition for quantitativedetermination of myo-inositol according to any one of claims 21 to 24,characterized in that the final concentration of thio-NAD is 2 to 10 mM.27. A method of eliminating glucose in a sample, which comprises atleast the steps of: reacting ATP with glucose in the sample to covertthem to ADP and glucose-6-phosphate; and reacting the thus obtained ADPwith glucose in the sample to covert them to AMP andglucose-6-phosphate.
 28. The method of detecting mild impaired glucosetolerance according to any one of claims 1 to 4, characterized in thatthe myo-inositol level is quantitatively determined after glucose in thesample is eliminated by a method comprising at least the steps of:reacting ATP with glucose in the sample to covert them to ADP andglucose-6-phosphate; and reacting the thus obtained ADP with glucose inthe sample to covert them to AMP and glucose-6-phosphate.